In vitro proliferation of hematopoietic stem cells in the absence of an adherent monolayer

Abstract

Experiments on long-term murine bone marrow cultures indicate that the production and maintenance of the hematopoietic stem cell is dependent on the establishment of an adherent monolayer and a secondary repopulation of the culture with fresh marrow. In contrast, we have found that bone marrow cultures derived from the Syrian hamster do not require a repopulation step and produce stem cells that proliferate and differentiate for more than 12 wk in the absence of an adherent layer. Stem cells were grown in Fisher's medium (pH 7.0–7.2) containing 20% horse serum in a fully humidified atmosphere of 5% CO2 in air at 37 degrees C. Cultures were fed twice weekly by removal of half of the medium and supernatant cells and replacement with an equal volume of fresh medium. No hormones or exogenous growth factors were required for the maintenance of myeloid cells, monocytes, and megakaryocytes in either the adherent or suspension cells cultures.

Bone marrow adipocytes support hematopoietic stem cell survival

“…Based on the well‐established ability of BM‐MSC to support hematopoietic cell growth via cytokine secretion (Eastment, Denholm, Katsnelson, & Arnold, ), BM‐A and BM‐MSC culture supernatants were examined for cytokines that might positively or negatively affect HSC support.…”

“…Since cell‐cell signaling is vital for HSC maintenance and activation (Eastment et al, ; Eaves & Eaves, ), five key pathways involved in the process were examined: activation of blood cells, leukocytes, phagocytes, myeloid cells, and antigen‐presenting cells. The five pathways exhibited differential regulation, and the cluster analysis findings obtained from array expression data demonstrated that BM‐A were closer to BM‐MSC than to AT‐A (Supplementary Figure S5A).…”

“…Hematopoiesis regulation in the BM niche is also mediated by stromal cell‐secreted factors, and cytokine and growth factor expression is critical for HSC maintenance and differentiation (Eastment et al, ). Remarkably, gene expression analysis showed the highest number of overexpressed cytokines in BM‐A, as 26 of the 30 genes studied were up‐regulated in BM‐A compared with AT‐A, and 20 were up‐regulated in BM‐A compared with BM‐MSC (Figure a,b).…”

“…Based on the well-established ability of BM-MSC to support hematopoietic cell growth via cytokine secretion (Eastment, Denholm, Katsnelson, & Arnold, 1982) (Supplementary Figure S2).…”

“…Since cell-cell signaling is vital for HSC maintenance and activation (Eastment et al, 1982;Eaves & Eaves, 1988), five key pathways involved in the process were examined: activation of blood cells, leukocytes, phagocytes, myeloid cells, and antigen-presenting cells.…”